EP3762503A1 - Séparation d'acides aminés basiques - Google Patents

Séparation d'acides aminés basiques

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Publication number
EP3762503A1
EP3762503A1 EP19763662.4A EP19763662A EP3762503A1 EP 3762503 A1 EP3762503 A1 EP 3762503A1 EP 19763662 A EP19763662 A EP 19763662A EP 3762503 A1 EP3762503 A1 EP 3762503A1
Authority
EP
European Patent Office
Prior art keywords
arginine
aqueous solution
amino acid
monophosphate
phosphate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19763662.4A
Other languages
German (de)
English (en)
Other versions
EP3762503A4 (fr
Inventor
Torgny NÄSHOLM
Jonathan LOVE
Mattias Holmlund
Nils Bertil SKOGLUND
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arevo AB
Original Assignee
Arevo AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Arevo AB filed Critical Arevo AB
Publication of EP3762503A1 publication Critical patent/EP3762503A1/fr
Publication of EP3762503A4 publication Critical patent/EP3762503A4/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05B—PHOSPHATIC FERTILISERS
    • C05B15/00—Organic phosphatic fertilisers
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
    • C05D9/00—Other inorganic fertilisers
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/10—Fertilisers containing plant vitamins or hormones
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G5/00—Fertilisers characterised by their form
    • C05G5/40—Fertilisers incorporated into a matrix
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/38—Separation; Purification; Stabilisation; Use of additives
    • C07C227/40—Separation; Purification
    • C07C227/42—Crystallisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C277/00—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C277/08—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups of substituted guanidines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05C—NITROGENOUS FERTILISERS
    • C05C11/00—Other nitrogenous fertilisers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia

Definitions

  • the present invention relates to the area of amino acid production. More specifically, the invention relates to a method of selectively separating basic amino acids from complex liquids including large number of different proteinaceous and/or nutritional components. Amino acids separated according to the invention are useful e.g. as slow release fertilizers of plants, as food ingredients or as feed additives. Background
  • Amino acids are organic compounds containing amine (-NH 2 ) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each amino acid.
  • the key elements of an amino acid are carbon (C), hydrogen (H), oxygen (O), and nitrogen (N), although other elements are found in the side chains of certain amino acids.
  • amino acids are important in nutrition and are commonly used in nutritional supplements, fertilizers, and food technology.
  • Industrial uses include the production of drugs, biodegradable plastics, and chiral catalysts.
  • US 4,006,004 (Seferian et al) relates to a phosphate enriched peat moss fertilizer. More specifically, a method for the production of free amino acid-containing phosphate- enriched high organic content fertilizers may include slurrying peat moss in up to five parts by weight of water per part of peat moss, mixing acid phosphate reactant solution of one part by weight of monopotassium acid phosphate with from one to four parts of orthophosphoric acid with the peat moss slurry in an amount of one to four parts by weight of the acid phosphate reactant per part of peat moss, heating the resulting mixture at about 90°C for not less than 4 hours to hydrolyze proteinaceous material in the peat moss into free amino acid, and recovering as product a peat moss fertilizer containing free amino acid, and enriched with phosphate and potassium.
  • the phosphoric acid reactant As the phosphoric acid reactant, orthophosphoric acid is preferred, particularly in its commercial form i.e. 85 percent H 3 P0 4 in aqueous solution.
  • the acid phosphate salt reactant may be an alkali metal i.e. potassium, sodium, lithium, or cesium metal, mono- or di- acid salt.
  • WO 2017/200468 SweTree Nutrition AB
  • fertilizer compositions comprising monophosphates of basic L-amino acids, such as arginine or lysine monophosphate.
  • the experimental part teaches how crystals of arginine monophosphate can be prepared from a supersaturated solution of arginine phosphate to which an equimolar amount of orthophosphorie acid was added.
  • the solution temperature wars increased to about 80°C, and the arginine phosphate solution was slowly cooled at a rate of approximately 5°C/hour. Crystal growth continued as the temperature decreased further. After reaching a temperature of 5°C, the remaining mother liquor was poured off.
  • the crude crystals were dried by vacuum filtration followed by drying in a heat cabinet at 35°C for approximately 24 hours.
  • US 9,682,026 (Colgate-Palmolive) relates to an oral care composition comprising arginine phosphate; sodium monofluorophosphate; and dicalcium phosphate dihydrate, which composition is substantially free of organic phosphates.
  • a method of producing such an oral care composition comprises the steps of combining together a basic amino acid component, a soluble fluoride salt and a calcium salt of an inorganic acid.
  • the basic amino acid is neutralized with an inorganic acid to form a salt of the basic amino acid prior to the combining step.
  • a number of separation steps are required in order to obtain a purified product.
  • EP 0 175 309 (Toray Industries, Inc) relates to L-lysine production, and more specifically to mutants which are capable of producing high yields in conventional fermentation processes.
  • the high yields are obtained due to the mutants being resistant to one or more a-substituted amino-epsilon-caprolactam compounds.
  • L-lysine is directly obtainable in the form of crystals from the medium.
  • One objective of the present invention is to provide an improved method for the manufacture of basic L-amino acids such as arginine or lysine by microbial
  • this objective may be achieved by a method of separating arginine or lysine from an aqueous solution, which method comprises at least the steps of
  • amino acid phosphate is selectively precipitated as a monophosphate of arginine or lysine.
  • Another objective of the present invention is to provide an efficient method for the production of phosphates of basic amino acids such as arginine or lysine from liquids that comprises amino acids and also have a complex content of proteinaceous components, such as proteins and/or protein decomposition products.
  • this may be achieved by a method of separating arginine or lysine from an aqueous solution, which method comprises at least the steps of
  • amino acid phosphate is selectively precipitated as a monophosphate of arginine or lysine.
  • Figure la, b and c are photographs of three different arginine monophosphate crystals derived according to the invention from different fermentation broths: Agrobacterium ( Figure la), E. coli ( Figure lb) and Corynebacterium ( Figure lc).
  • Figure 2a, b and c are gas chromatograms of monophosphates produced according to the invention, as described in the experimental part below.
  • Figure 3 a and b are chromatograms of a) a modified fermentation broth, and b) solubilized arginine phosphate crystals derived from the modified fermentation broth, respectively.
  • Figure 4a and b illustrate the reaction time of a method according to the invention when acid is added to base.
  • Figure 5 a and b illustrate the reaction time of a method according to the invention when base is added to acid.
  • Figure 6 illustrates the energy used for heating of water in the two methods of Figure 5 and 6, respectively.
  • Figure 7a and b illustrate the arginine recovery obtainable from raw E.coli
  • the present invention relates to a general method of separating basic amino acids such as arginine or lysine from an aqueous solution, which method comprises at least the steps of
  • amino acid phosphate is selectively precipitated as a monophosphate of arginine or lysine.
  • the amino acid may be selected from the group consisting of arginine, lysine and histidine.
  • the amino acid may be an L-amino acid, such as L-arginine or L-lysine, which is the form most advantageously used e.g. in plant fertilizers which is also produced by cultured bacteria.
  • the phosphate is an arginine monophosphate
  • the phosphoric acid is then added at an approximately equimolar amount of 1 : 1 to the concentration of arginine in the solution provided in step a).
  • Using different molar ratios of phosphoric acid:amino acid may be used to obtain other phosphates than the monophosphate.
  • step b) the combination of phosphoric acid with the aqueous solution may be performed as traditionally advised when acids are used by following the principle of “acid to base”, i.e. by titering the phosphoric acid into the aqueous solution.
  • acids are used by following the principle of “acid to base”, i.e. by titering the phosphoric acid into the aqueous solution.
  • the present inventors have surprisingly found that by using the reverse procedure, i.e. by titering the aqueous solution into the phosphoric acid, crystals of unexpectedly high purity could rapidly be obtained. This is described further in Example 5 below.
  • the heat generated in the resulting exothermic reaction could be utilized instead of external heating, providing a very efficient process in terms of resources.
  • the pH will be set to an appropriate value depending on the specific amino acid separated.
  • the method may be a multistep process where firstly, one amino acid is selectively precipitated as its monophosphate at a first pH, and secondly, the solution is adjusted to a second pH at which a second amino acid is selectively precipitated.
  • the invention may be utilized to obtain a highly purified amino acid, in which case step e) is performed to redissolve the precipitated amino acid phosphate after removal thereof from the original solution, followed by removal of the phosphate by any conventional method such as liquid chromatography or precipitation.
  • the present invention may be utilized to obtain a phosphate such as a monophosphate of arginine or lysine, which find subsequent use e.g. as plant fertilizers.
  • the present inventors have found that the above described method of precipitation may successfully be followed by one or more steps of adding at least one zeolite to the aqueous solution from which arginine or lysine
  • the present invention also relates to a method of recovering basic amino acids such as arginine or lysine from a complex solution, which method comprises at least the steps of
  • step d) combining the aqueous solution separated in step d) with at least one zeolite under conditions allowing for adsorption of arginine or lysine to the zeolite(s).
  • the conditions for adsorption of basic L-amino acids to zeolites has been described in WO 2017/222464, from which the skilled person may use teachings with regard to the zeolite step(s) in the design of a multistep process wherein a precipitation of arginine is followed by adsorption of remaining arginine or lysine to at least one zeolite having the appropriate properties. Additional zeolite adsorption steps may be performed on any liquid resulting from step f), optionally using zeolite(s) having properties different from those used in the preceding adsorption step.
  • the aqueous solution provided in step a) may be a broth originating from the fermentation of cells producing proteins, peptides and/or amino acids in an undefined growth medium, such as LB medium or any other composition.
  • a medium will commonly include a large number of components such as sugars and nutrients added for growth as well as various other products expressed by the cells, which will require removal by separate purification steps in the conventional biotechnological manufacture of amino acids.
  • the method according to the invention will include step e) wherein the amino acid phosphate precipitation is redissolved to separate the amino acid from the phosphate.
  • the cells are bacterial strains selected from the group consisting of Escherichia, Corynebacterium, and Agrobacterium.
  • the currently available methods for separating amino acids from fermentation broths are resource-demanding multistep methods for purification such as several liquid chromatography steps which may be avoided and replaced by the precipitation as phosphate according to the invention.
  • the present aspect enables more efficient production of amino acids than conventionally used purification protocols.
  • this aspect may be used to produce amino acid phosphates for subsequent use in the manufacture of plant fertilizers.
  • amino acids are common products in most protein expression
  • this aspect allows for an efficient utilization of broths from which e.g. proteins have been recovered as the primary targets.
  • the aqueous solution provided in step a) is a liquid originating from hydrolysis of animal or vegetable matter.
  • a source of commercial interest is a solution originating from the hydrolysis of feather.
  • Feathers are readily available as by-product from poultry slaughterhouses, and contain 85% - 90% keratin. Hydrolysis of keratin is frequently used, wherein the disulphide bonds and peptide bonds are broken to form smaller proteins, peptides and amino acids.
  • the conventional industrial feather protein processing method is thermal pressure hydrolysis, which requires further processing using chromatography and other purification techniques to obtain amino acids of high purity.
  • the present invention presents an advantageous alternative to recover amino acids from the feather industry.
  • the aqueous solution provided in step a) is a rest stream originating from pulp manufacture.
  • pulp is used herein for a
  • lignocellulosic fibrous material prepared by chemically or mechanically separating cellulose fibres from wood, fibre crops, waste paper, or rags.
  • the rest streams originating from pulp mills are highly complex solutions which may include lignin and cellulose as well as various chemicals.
  • a preceding step of filtering the solution may be included.
  • highly purified basic amino acids or as well as amino acid monophosphates may be obtained from this solution.
  • the method according to the invention may be used to produce a basic amino acid phosphate such as arginine monophosphate or lysine monophosphate which is subsequently combined with one or more nutrients to form a plant fertilizer.
  • the method according to the invention may also include a step of granulating the precipitated arginine or lysine monophosphate to obtain a format suitable for its use in solid fertilizer compositions. Further, the method according to the invention may comprise a step of formulating a fertilizer by combining arginine or lysine monophosphate precipitated as described above with at least one binder and optionally one or more additional nutrients to form a plant fertilizer. Binders suitable for the use with arginine or lysine monophosphate in fertilizer compositions are discussed in WO 2017/200468.
  • the present invention also relates to a fertilizer composition which includes arginine or lysine monophosphate precipitated as described above, optionally in combination with at least one binder and/or one or more additional nutrients.
  • a fertilizer may be a solid composition, such as a granulate.
  • the invention relates to a solid fertilizer composition which comprises arginine or lysine monophosphate, optionally in a granulate with a suitable binder, and at least one zeolite to which arginine or lysine has been adsorbed.
  • the relative proportions of the two components may be varied and decided for each specific formulation based on the desired properties of the fertilizer, such as release rate.
  • Figure 1 is a photograph of three arginine monophosphate crystal derived according to the invention from three different fermentation broths: Agrobacterium ( Figure la) and E. coli ( Figure lb) spiked with lOOg/L of arginine to simulate broths from arginine- overproducing strains; as well as a modified fermentation broth from arginine- overproducing Corynebacterium ( Figure lc) with an arginine concentration of 300g/L treated with an equimolar amount of phosphoric acid (1.72 mole arginine/ 1.72 mole phosphoric acid) and left at room temperature for 5 days. Arginine monophosphate crystals were formed during this period and retrieved from the solution. In this example, 75% orthophosphoric acid was used. As appears from visual inspection, the crystals have a very high purity.
  • Figure 2 shows gas chromatograms obtained by performing gas chromatography following well established protocols on monophosphates produced according to the invention, as described in the experimental part below.
  • Figure 2a is a gas chromatogram of 1) a pure arginine reference, 2) a modified fermentation broth. Arginine peaks were masked at 412-422 s, 492-497 s and 540-555 s in order to increase sensitivity to non-arginine content. A peak at 597 s is derived from the pure arginine reference. Numerous peaks (for example at 323 s,
  • chromatogram 2 365 s, 391 s, 395 s, 400 s, 432 s, 434 s, 473 s, 5 l0s, 519 s, 523 s, 525 s, 527 s, 535 s, 575 s, 575 s, 579 s, 581 s, 583 s and 587 s) in chromatogram 2 are reduced in amplitude or not evident in chromatogram 1 indicating an expected composition complexity in the fermentation broth not present in the pure arginine reference.
  • Figure 2b is a gas chromatogram of 1) a pure arginine reference solution and 2) arginine monophosphate crystals derived from the modified fermentation broth by way of the method described herein. Arginine peaks were masked at 412-422 s, 492-497 s and 540-555 s in order to increase sensitivity to non-arginine content. The phosphate content of the crystals is evident in chromatogram 2 at ca. 421 s. Besides the extra peak from phosphate the two chromatograms are very similar indicating arginine phosphate crystals have a high level of purity.
  • Figure 2c is a gas chromatogram of 1) crystals precipitated from solution made from arginine phosphate crystals derived from the fermentation broth vs 2) a modified fermentation broth solution. Arginine peaks were masked at 412-422 s, 492-497 s and 540-555 s in order to increase sensitivity to non-arginine content. The phosphate content of the crystals is evident in chromatogram 3 at ca. 421 s.
  • peaks for example at 323 s, 365 s, 391 s, 395 s, 400 s, 432 s, 434 s, 473 s, 5 l0s, 519 s, 523 s, 525 s, 527 s, 535 s, 575 s, 575 s, 579 s, 581 s, 583 s and 587 s
  • Figure 3 are chromatograms of a) a modified fermentation broth, and b) solubilized arginine phosphate crystals derived from the modified fermentation broth,
  • the peak at 1.6 min represents derivation residues, 2.2 ammonium, 2.8 arginine, 3.1 glycine, 3.9 glutamic acid, 4.1 threonine,
  • Figure 4 is an illustration of the reaction time of a method according to the invention when acid is added to base. More specifically, Figure 4a illustrates Method 1, wherein arginine base was dissolved in 500 ml, 60 °C dH 2 0. The arginine solution was stirred and 75% phosphoric acid (giving an arginine to phosphate ratio of 1: 1) was titered into the arginine solution at a rate in which the temperature was not allowed to exceed 60 °C ( Figure 4b). The vertical dotted line in Figure 4a indicates the time for full mixing of arginine and phosphoric acid.
  • Figure 5 is an illustration of the reaction time of a method according to the invention when base is added to acid. More specifically, Figure 5a illustrates Method 2, wherein a slurry of only partially dissolved arginine base in 15°C dH 2 0 (62.5 g dissolved and 187.5 g as an undissolved. The arginine solution was titered into a solution of 75% phosphoric acid with stirring at a rate so that the temperature was not allowed to exceed 60 °C until complete mixing of arginine and phosphoric acid was achieved (Figure 5b). The vertical dotted line in Figure 5a indicates the time for full mixing of arginine and phosphoric acid.
  • Figure 6 illustrates the energy used for heating of water in the two methods of Figure 5 and 6, respectively.
  • Method 1 which utilizes the traditional sequence of“add acid to water (Add the Acid)”, uses almost 200 kJoule/litre; while the energy consumption of the less frequently used sequence of“add water to acid” is close to zero thanks to the utilization of the exothermic heat.
  • Figure 7 illustrates the arginine recovery obtainable from raw E.coli fermentation broth following the method described in Example 6, i.e. repeated steps of recovery through crystallization followed by addition of zeolites. More specifically, Figure 7a shows the arginine concentration remaining in raw fermentation broth (% of initial solution concentration corresponding to 140 g arginine/1) after arginine crystallization using phosphoric acid (1), treatment of fermentation broth using zeolites (2) and repeated treatment of fermentation broth using zeolites (3).
  • Figure 7a illustrates the recovery of arginine as described above, i.e. from raw fermentation broth (% of initial amount corresponding to 140 g arginine/1) after arginine crystallization using phosphoric acid (1), treatment of fermentation broth using zeolites (2) and repeated treatment of fermentation broth using zeolites (3).
  • a fermentation broth produced from an arginine-overproducing Corynebacterium with an arginine concentration of 300 g/L was treated with an equimolar amount (1.72 mole arginine/ 1.72 mole phosphoric acid) of phosphoric acid (75% orthophosphoric acid) and left at room temperature for 5 days.
  • Arginine monophosphate crystals were formed during this period and retrieved from the solution. The crystals were analysed with Gas Chromatography-Mass Spectrometry for contaminants of other compounds other than arginine and phosphate and the impurities were found to comprise less than 0.01 % of the total mass.
  • a fermentation broth with E.coli was spiked with arginine to a concentration of 100 g/L to simulate an arginine-overproducing strain of the bacteria.
  • the arginine-spiked broth was treated with equimolar amounts (0.57 mole arginine/ 0.57 mole phosphoric acid) of phosphoric acid (75% orthophosphoric acid) and left at room temperature for 5 days.
  • Arginine phosphate crystals were formed during this period and retrieved from the solution. The crystals were analysed with Gas Chromatography-Mass
  • a fermentation broth with Agrobacterium was spiked with arginine to a concentration of 100 g/L to simulate a arginine overproducing strain of the bacteria.
  • the arginine spiked broth was treated with equimolar amounts (0.57 mole arginine/ 0.57 mole phosphoric acid) of phosphoric acid (75% orthophosphoric acid) and left at room temperature for 5 days.
  • Arginine phosphate crystals were formed during this period and retrieved from the solution. The crystals were analysed with Gas Chromatography- Mass Spectrometry for contaminates of other compounds than arginine and phosphate and the impurities were found to comprise less than 0.01 % of the total mass.
  • Figure 1 A fermentation broth with Agrobacterium was spiked with arginine to a concentration of 100 g/L to simulate a arginine overproducing strain of the bacteria.
  • the arginine spiked broth was treated with equimolar amounts (0.57 mole arginine/ 0.57 mo
  • Example 4 A fermentation broth with E.coli was spiked with L-arginine to a concentration of 140 g/L to simulate a fermentation broth from an arginine overproducing strain of the bacteria.
  • the arginine spiked broth was treated with equimolar amounts of phosphoric acid (0.80 mole arginine/ 0.80 mole phosphoric acid) and left at room temperature for 5 days.
  • Arginine phosphate crystals were formed during this period and retrieved from the solution.
  • the modified fermentation broth and crystals was analysed with Ultra Performance Liquid Chromatography for ammonium and amino acid composition.
  • Arginine phosphate crystals were produced from complex solutions, and the two alternative procedures of the invention were compared.
  • arginine base (CAS: 74-79-3) was dissolved in 500 ml, 60 °C dH 2 0.
  • the arginine solution was stirred and 119 ml of 75% phosphoric acid (arginine/phosphate 1: 1) was titered into the arginine solution at a rate were the temperature was not allowed to exceed 60 °C. It is well known that heating of arginine solutions above 60 °C will lead to the breakdown of arginine into ornithine and ammonium.
  • the time for completing the mixing of arginine and phosphoric acid was measured, see Figure 4a and b.
  • the alternative procedure sometimes referred to as a“base to acid” method, was set up as follows: 250 g of arginine base was partly dissolved in 15 °C dH 2 0 62.5 g dissolved and 187.5 g as an undissolved. Instead of titering the acid into the base, a reverse procedure was used in which the arginine slurry was titered into 119 ml of 75% phosphoric acid with stirring at a rate were the temperature was not allowed to exceed 60 °C.
  • the rate of phosphoric acid incorporation will be strictly limited by the exotherm that risks leading to degradation of arginine if the temperature exceeds 60 °C.
  • the energy requirement in the first procedure was 190 kJoule/1 for heating water from 15 to 60 °C, see Figure 6, Method 1.
  • energy for heating of the arginine solution was provided by the exothermal reaction when the arginine slurry was added into phosphoric acid, and hence no energy for heating was required, see Figure 6, Method 2.
  • the present example was set up to illustrate how the present invention may be utilized in process to produce pure arginine phosphate crystals from complex solutions with high recovery.
  • Arginine was incorporated into zeolites as follows: 21.5 g of Clinoptilolite natural zeolite 1-3 mm (CAS: 12173-10-3) was loaded with arginine from 100 ml E.coli fermentation broth containing 52.5 g/1 arginine. The solutions remaining after zeolite treatment were analysed on an UPLC for arginine concentration. The treatment was repeated 2 times.
  • Amino acids were determined after separation on an AccQ-Tag Ultra column by elution with a mixture of 99.9% formic acid and 10% acetonitrile at the following gradient 0-5.74 min isocratic 99.9% formic acid, declining to 90.9% formic acid from 5.74 min to 7.74 min, to 78.8% formic acid at 8.24 min and then to 40.4% formic acid at 8.74 min, before re-equilibration with 99.9% formic acid from 8.74 to 9.54 min.
  • the flow rate was 0.6 ml/min and the column temperature was set to 55 °C.
  • a fermentation broth with E.coli was spiked with arginine to a concentration of 140 g/L to simulate a arginine overproducing strain of the bacteria.
  • the arginine spiked broth was treated with equimolar amounts of phosphoric acid (0.80 mole arginine/
  • the arginine concentration was measured after the zeolite addition and the arginine concentration in the fermentation broth was found to be 37.5 g/L.
  • the zeolite treatment was repeated resulting in an arginine concentration of 22.5 g/L in the fermentation broth.
  • the total recovery of arginine from the fermentation broth after these steps was 84%, see Ligure 7a and b.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Fertilizers (AREA)

Abstract

La présente invention concerne une méthode de séparation d'acides aminés à partir d'une solution aqueuse comprenant des acides aminés ainsi que des protéines et/ou des produits de décomposition de protéines par combinaison d'acide phosphorique avec la solution aqueuse pour précipiter un monophosphate d'acide aminé. Le précipité ainsi formé peut être séparé de la solution aqueuse et utilisé en tant que tel, par exemple dans des compositions d'engrais végétaux. Le précipité peut être redissous dans un liquide approprié, après quoi le phosphate et l'acide aminé peuvent être séparés selon des méthodes classiques. Dans les deux cas, la présente invention permet la précipitation hautement sélective de monophosphate d'arginine et/ou de lysine sous forme de cristaux.
EP19763662.4A 2018-03-05 2019-03-05 Séparation d'acides aminés basiques Pending EP3762503A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE1850233A SE543703C2 (en) 2018-03-05 2018-03-05 Separation of basic amino acids
PCT/SE2019/050188 WO2019172825A1 (fr) 2018-03-05 2019-03-05 Séparation d'acides aminés basiques

Publications (2)

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EP3762503A1 true EP3762503A1 (fr) 2021-01-13
EP3762503A4 EP3762503A4 (fr) 2021-12-22

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EP19763662.4A Pending EP3762503A4 (fr) 2018-03-05 2019-03-05 Séparation d'acides aminés basiques

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US (1) US11814334B2 (fr)
EP (1) EP3762503A4 (fr)
JP (1) JP7503496B2 (fr)
KR (1) KR20200126981A (fr)
SE (1) SE543703C2 (fr)
WO (1) WO2019172825A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2020380098A1 (en) * 2019-11-06 2022-04-28 Arevo Ab Preparations for enhanced biocontrol

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS536236B1 (fr) * 1969-02-03 1978-03-06
US4006004A (en) 1975-08-25 1977-02-01 Rupen B. Seferian Phosphate enriched peat moss fertilizer and method therefor including free amino acid supplementation
JPS6087277A (ja) * 1983-10-19 1985-05-16 Taisho Pharmaceut Co Ltd 光学活性トランス−エポキシコハク酸ジアルキルエステルの製造法
EP0175309A3 (fr) * 1984-09-14 1987-11-11 Toray Industries, Inc. Procédé de préparation de L-lysine
US5177069A (en) * 1989-12-13 1993-01-05 Kissei Pharmaceutical Co., Ltd. Naphthysulfonylalkanoic acid compounds and pharmaceutical compositions thereof
DE4217203C2 (de) * 1992-05-23 1995-09-21 Degussa Verfahren zum Abtrennen von Aminosäuren aus wäßrigen Lösungen
WO2005123669A1 (fr) 2004-06-10 2005-12-29 Board Of Trustees Of Michigan State University Synthese du caprolactam a partir de la lysine
KR100733928B1 (ko) * 2005-11-30 2007-07-02 씨제이 주식회사 카나마이신 내성을 갖고 ｌ-라이신 생산능이 향상된코리네형 미생물 및 그를 이용하여 ｌ-라이신을 생산하는방법
JP4915723B2 (ja) * 2006-03-13 2012-04-11 コスモ石油株式会社 アミノ酸リン酸類塩の製造方法
BRPI0703692B1 (pt) 2006-12-25 2016-12-27 Ajinomoto Kk método para se obter os cristais de um hidrocloreto de aminoácido básico compreendendo gerar um aminoácido básico usando células microbianas por fermentação em um caldo de fermentação ou por um método enzimático em uma solução de reação de enzima usando as células como catalisadores
CN104887538B (zh) 2008-02-08 2019-03-29 高露洁-棕榄公司 口腔护理产品及其使用和制造方法
BR112018071850B1 (pt) * 2016-05-16 2023-10-24 Arevo Ab Fertilizante, composição fertilizante e método de aumento do crescimento de uma planta
CA3028342C (fr) * 2016-06-23 2024-06-04 Arevo Ab Composition d'engrais comprenant une zeolite et un acide l-amine basique

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JP7503496B2 (ja) 2024-06-20
EP3762503A4 (fr) 2021-12-22
US11814334B2 (en) 2023-11-14
SE1850233A1 (en) 2019-09-06
KR20200126981A (ko) 2020-11-09
US20210017122A1 (en) 2021-01-21
JP2021515551A (ja) 2021-06-24
SE543703C2 (en) 2021-06-15
WO2019172825A1 (fr) 2019-09-12

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